Pin1/YAP pathway mediates matrix stiffness‐induced epithelial–mesenchymal transition driving cervical cancer metastasis via a non‐Hippo mechanism

Abstract Cervical cancer metastasis is an important cause of death in cervical cancer. Previous studies have shown that epithelial–mesenchymal transition (EMT) of tumors promotes its invasive and metastatic capacity. Alterations in the extracellular matrix (ECM) and mechanical signaling are closely associated with cancer cell metastasis. However, it is unclear how matrix stiffness as an independent cue triggers EMT and promotes cervical cancer metastasis. Using collagen‐coated polyacrylamide hydrogel models and animal models, we investigated the effect of matrix stiffness on EMT and metastasis in cervical cancer. Our data showed that high matrix stiffness promotes EMT and migration of cervical cancer hela cell lines in vitro and in vivo. Notably, we found that matrix stiffness regulates yes‐associated protein (YAP) activity via PPIase non‐mitotic a‐interaction 1 (Pin1) with a non‐Hippo mechanism. These data indicate that matrix stiffness of the tumor microenvironment positively regulates EMT in cervical cancer through the Pin1/YAP pathway, and this study deepens our understanding of cervical cancer biomechanics and may provide new ideas for the treatment of cervical cancer.

process in which epithelial cells are transformed into mesenchymal phenotype cells through a specific procedure, mainly manifested by decreased expression of epithelial markers such as Ecadherin and enhanced expression of mesenchymal markers such as Vimentin, leading to enhanced cell migration. 5 Recent studies have shown that EMT is not a binary process but a dynamic process in which cells in different stages of the process can coexist in the same tumor. [6][7][8][9] Importantly, cancer cells in a partial EMT state exhibit a more aggressive phenotype than those in a complete EMT state. 10 Tumor metastasis is often closely related to the alteration of tumor microenvironment. [11][12][13][14] As an important component of the tumor microenvironment, extracellular matrix (ECM) stiffness not only maintains the three-dimensional morphological structure of tumor tissue but also generates biochemical or biophysical signals that affect the biological functions of tumor cells. [15][16][17] A study found that in cervical cancer, the involvement of cancer-associated fibroblasts (CAF) in the remodeling of the ECM led to an increase in the stiffness of tumor tissue and promoted the progression of cervical cancer. 18 In addition, higher matrix stiffness promotes cervical cancer cell proliferation and chemoresistance. 19 However, past studies have focused on the role of various extracellular and intracellular biochemical signals in tumor progression, 20,21 with little knowledge on how matrix stiffness, simply as an independent cue, initiates EMT in cervical cancer cells.
Therefore, we aimed to investigate whether and how tissue mechanics affects EMT in cervical cancer. In this study, we elucidated the molecular mechanism by which matrix stiffness alone acts as an initiator to induce EMT in cervical carcinoma based on an in vitro collagen-coated polyacrylamide (PA) hydrogel model with adjustable stiffness and an animal model. We verified the effect of matrix stiffness on EMT in cervical cancer and the role of yes-associated protein (YAP) and PPIase non-mitotic a-interaction 1 (Pin1) in it. We further found that matrix stiffness regulates YAP activity through Pin1 in a non-Hippo pathway. The present work highlights the important role of biomechanical signaling in triggering EMT and promoting invasive metastasis in cervical cancer, providing a promising idea for matrix stiffness therapy for the prevention of cervical cancer metastasis.

| Matrix stiffness induces the occurrence of EMT in cervical cancer
Increased surface tension and stiffness of tumor cells have been reported to make them more biologically invasive. 17 To investigate whether matrix stiffness can regulate the biological behavior of cervical cancer, we prepared a collagen-coated polyacrylamide hydrogel (PA gel) system with Young's modulus of 1 and 20 kPa to simulate soft and stiff matrix in vitro. 22 Under the microscope, significant differences in cell morphology were observed when hela cells were inoculated on a stiff and soft hydrogel. Cells on the soft matrix are round in shape, with round clusters of cells, whereas cells on the stiff matrix are elongated (Figure 1a) and significantly larger surface areas in response to stiff matrix (Figure 1b). In addition, F-actin remodeling was observed in cells cultured on stiff matrix (Figure 1c), indicating that stiffness may influence cell motility. Compared with cells cultured on soft matrix, cells cultured on stiff matrix had a greater ability to migrate and proliferate (Figure 1d,e), indicating that matrix stiffness may influence malignant behavior of hela cells.
In the process of tumor metastasis, EMT often occur to enhance its invasions, among which the downregulation of epithelial marker Ecadherin and the activation of mesenchymal marker Vimentin are regarded as the marker changes of EMT. 23 Western blot and quantitative PCR (qPCR) results showed that Ecadherin expression was reduced and Vimentin expression was increased in stiff matrix cells compared to soft matrix, and these results showed that matrix stiffness stimulation induced the occurrence of EMT in hela cells (Figure 1f and Figure S1). Interestingly, the induced EMT changes and associated signaling changes were reversed when cells initially cultured on stiff and re-cultured on soft matrix ( Figure S2A,B). To investigate the EMT status of cells undergoing matrix stiffness stimulation, we used immunofluorescence staining to observe the expression of E-cadherin and Vimentin in cells cultured on soft and stiff matrix. The results showed a significant increase in Vimentin expression and decrease in E-cadherin expression in cells cultured on stiff matrix compared to soft matrix, but cells on stiff matrix still expressed a small amount of E-cadherin protein, which shows a presence of co-expression of E-cadherin and Vimentin in cells ( Figure 1g), suggesting that cells undergoing matrix stiffness stimulation are experiencing a partial EMT state, which is consistent with the performance described in previous studies. 24,25 In order to further study the effect of matrix stiffness to the hela cells in vivo, we established a cell-derived xenograft model and applied F I G U R E 1 Matrix stiffness induces the occurrence of epithelial-mesenchymal transition (EMT) in cervical cancer. (a) Microscopy showing the typical morphology of hela cells cultured on soft and stiff matrix and quantification of cell long-to-short axis (***p < 0.001), scale bar: 50 μm. (b) Cell surface area was calculated by phase-contrast analysis of cell images on soft and stiff matrix (***p < 0.001). (c) Immunofluorescence images of hela cells cultured on soft and stiff matrix and quantified for phalloidin (green) and 4',6-diamidin0-2-phenylindole (DAPI) (blue) (**p < 0.01), scale bar: 10 μm. (d) Migration trajectory and quantification of hela cells cultured on soft or stiff matrix for 24 h (h) (**p < 0.01), scale bar: 100 μm. (e) Western blot analysis of proliferating cell nuclear antigen (PCNA) in hela cell, cultured on soft and stiff matrix showed expression and quantification. β-Actin was used as a control (***p < 0.001). (f ) Western blot analysis of E-cadherin and Vimentin in hela cell, cultured on soft and stiff matrix showed expression and quantification, GAPDH was used as a control (***p < 0.001). (g) Immunofluorescence images of Vimentin (red), E-cadherin (green) and DAPI (blue) in hela cells cultured on soft and stiff matrix, scale bar: 10 μm.

| Matrix stiffness modulates EMT in cervical cancer by promoting YAP nuclear localization
YAP is widely present in vivo and functions as a sensor and mediator of mechanical signals indicative of the cellular microenvironment. 28 As a typical mechanism of YAP regulation, the phosphorylation cascade reaction of the Hippo pathway has been identified. 29 The Hippo pathway phosphorylates YAP through large tumor suppressor (LATS), causing it to stay in the cytoplasm to inhibit its activity, while dephosphorylated YAP enters the nucleus with the transcriptional enhancer subdomain (TEAD), regulating the transcription of specific genes thereby promoting cell proliferation and survival. 30,31 As YAP activation in many tumors can lead to EMT, 32

| Matrix stiffness modulates EMT in cervical cancer by modulating Pin1 activity
Pin1 is a member of the peptidyl prolyl isomerase (PPIase) family 34 that specifically binds proteins containing phosphorylated Ser/Thr-Pro motifs that alters the function of phosphorylated proteins to regulate signal transduction after phosphorylation, which plays an important role in cancer and neuropathy. 35,36 Inhibition of Pin1 has been reported to significantly reduce the proliferation and metastasis of cervical cancer cells, and it may serve as a new therapeutic target for cervical cancer. 37 To investigate whether Pin1 is involved in the regulation of EMT in hela

| Matrix stiffness modulates YAP nuclear translocation via Pin1 in a non-Hippo pathway
The current study shows that Pin1, as a positive regulator of YAP, can increase the activity and stability of YAP. 41 In addition, Pin1 interacts with STK3 (MST2), an upstream regulator of the Hippo pathway, to regulate STK3 activity, which in turn promotes YAP entry into the nucleus and exerts biological effects. 42 Thus, we investigated whether Pin1 mediated the activation of matrix stiffness regulated YAP. First, we overexpressed Pin1 in soft matrix cultured cells using expression plasmids and inhibited Pin1 in stiff matrix cultured cells using Juglone.
By immunostaining and Westernblot analysis of nuclear and cytoplasmic, we found that Pin1 overexpression promoted YAP nuclear localization in soft matrix cultured cells, while Pin1 inhibition decreased YAP nuclear localization in stiff matrix cultured cells (Figure 4a-c). To resolve the upstream-downstream relationship between YAP and Pin1, we performed reverse experiments. First, we used a YAP expression plasmid to overexpress YAP in cells cultured on soft and stiff matrix and found that overexpression of YAP did not alter the expression of Pin1 (Figure 4d). We also treated hela cells transfected with the Pin1 expression plasmid using the YAP inhibitor verteporfin.
The results show that verteporfin reverses the cellular EMT alterations promoted by Pin1 overexpression (Figure 4e). Taken together, our results indicate that matrix stiffness can promote YAP nuclear translocation via Pin1.
The phosphorylation cascade of Hippo pathway modulates the phosphorylation of YAP is currently the classical theory to explain the cause of YAP nuclear translocation. 43 Among them, LATS1, as one of the cores of Hippo pathway, plays a key role in the phosphorylation of YAP by Hippo pathway. 44 To investigate whether Pin1 affects YAP via Hippo pathway kinase under matrix stiffness stimulation, we first performed western blotting assays on LATS1 protein and found that neither Pin1 overexpression nor inhibition treatment altered LATS1 expression (Figure 5a F I G U R E 5 Matrix stiffness regulates YAP nuclear translocation via Pin1 in a non-Hippo pathway. (a) Western blot analysis of LATS1 in hela cell treated with empty vector and Pin1 plasmid showed expression and quantification. GAPDH was used as control ("ns" means no significance). (b) Western blot analysis of LATS1 in hela cell treated with DMSO and Juglone showed expression and quantification. GAPDH was used as a control ("ns" means no significance). (c) Western blot analysis and quantification of YAP, p-YAP in cells overexpressing Pin1 on soft matrix. GAPDH and YAP was used as a control ("ns" means no significance, *p < 0.05). (d) Western blot analysis and quantification of YAP, p-YAP in cells inhibiting Pin1 cultured on stiff matrix. GAPDH and YAP was used as a control ("ns" means no significance, **p < 0.01). (e) Immunofluorescence images and quantification of Cofilin (red), phalloidin (green) and DAPI (blue) in hela cells overexpressing Pin1 cultured on soft matrix, scale bar: 10 μm (***p < 0.001). (f) Immunofluorescence images and quantification of Cofilin (red), phalloidin (green) and DAPI (blue) in hela cells inhibited Pin1 cultured on stiff matrix, scale bar: 10 μm (***p < 0.001). (g) Western blot analysis and quantification of Cofilin in cells overexpressing Pin1 on soft matrix. GAPDH was used as a control (***p < 0.001). (h) Western blot analysis and quantification of Cofilin on cells inhibiting Pin1 on stiff matrix. GAPDH was used as a control (***p < 0.001). study 47 indicates that remodeling of F-actin can directly regulate YAP activity through non-Hippo pathway pathways. In the preceding Taken together, we reveal that Pin1 regulates YAP nuclear translocation through F-actin remodeling independent of Hippo pathway kinase.

| Pin1 modulates EMT in cervical cancer in vivo
To further investigate the role of Pin1 in hela cell EMT in vivo, we

| DISCUSSION
In this study, we demonstrated that matrix stiffness induces the occurrence of EMT in cervical cancer. Notably, we found that the activity of Pin1 was able to change with the stimulation of matrix stiffness. More importantly, we found that matrix stiffness regulates YAP nuclear translocation via Pin1 in a non-Hippo pathway thus promote EMT in cervical cancer. This study provides evidence that targeting ECM stiffness can hinder EMT in cervical cancer and indicates that Pin1 and YAP may be a promising target for treatment against cervical cancer.
The EMT program is modulated by the network of multiple pro-EMT factors such as TWIST1. Recent studies showed that matrix stiffness drives EMT and tumor metastasis through a TWIST mechanotransduction pathway. 24,48 They also noticed matrix rigidity triggers YAP nuclear localization in breast cancer. However, there are distinct differences between the mechanotransduction pathways of YAP/TAZ and TWIST1. TWIST1 are only sensitive in matrix stiffness, while YAP/TAZ also make response to the cellular polarity, shape and adherens junction. Besides, they treated MCF10A cells with leptomycin B, a nuclear export inhibitor, and found that YAP accumulated into the nucleus on compliant matrix whereas TWIST1 not. These researches mainly focus on molecular pathway of TWIST1 signaling axis, the YAP/TAZ mechanotransduction pathway remains to be elucidated.
The present study shows that Pin1 activity varied with matrix stiffness and was able to regulate F-actin remodeling, probably because Pin1 expression increased Cofilin activity, thus regulating F-actin remodeling. More importantly, we found that matrix stiffness regulates YAP nuclear translocation via Pin1 in a non-Hippo pathway thus promoting EMT in cervical cancer. We found that Pin1 regulates YAP nuclear translocation in a nonphosphorylation-dependent manner, the main reason is quite likely the cytosolic pore opening caused by skeletal alterations which in turn leads to direct entry of YAP into the nucleus. 49,50 But all of the altered import and export rates caused by nuclear pore opening experimental phenomena have not been explained by a reasonable model, 45 this remains to be investigated.
Multiple recent reports showed EMT and matrix stiffness have reciprocal crosstalk on each other. A previous study showed that lung cancer cells that have undergone EMT can activate the FAK/SRC axis by altering the collagen composition of their surrounding microenvironment, leading to invasion and metastasis. 51 Their study also revealed that miR-200 and ZEB1 can affect the migration ability of mesenchymal lung cancer cells in vitro through direct regulation of LOX proteins. A recent study also demonstrated that cancer cells can maintain the stability of the mesenchymal state of the cell through the ZEB1-LOXL2-ECM circuit and make the return to the epithelial phenotype more difficult, which has important implications for the distant metastasis of cancer cells. 52 These results suggest that LOX may serve as a potential clinical therapeutic target. Gibbons's team 51 used BAPN drugs to inhibit LOX enzyme activity, and transwell migration and collagen invasion assays found that lung cancer cell migration was inhibited in vitro, while in mice vivo experiments did not show significant change in tumor size. In our study, inhibition of ECM stiffness using BAPN significantly impeded the ability of metastasis in cervical cancer, and these results are consistent with several studies confirming the effectiveness of chemical LOX inhibitors in vivo. 27,[53][54][55][56] Their results are not consistent with ours, which may be due to an off-target effect. They also explored the consideration of whether there is a link between LOX and YAP activity, but this remains to be further investigated.
It is worth to note not all tumor types are highly sensitive to the ECM stiffness stimulation. 15,57 The crosstalk of ECM stiffness and tumor cells has been well studied in adenocarcinoma such as breast cancer in contrast to squamous cell carcinoma. Interestingly, there might be differences exist between carcinoma types. Mammary epithelial cells initially cultured on stiff and re-cultured on soft matrix, exhibit active migration behaviors after re-cultured on soft matrix. 58 This phenomenon is defined as cellular memory, meaning that past exposure to different values of matrix stiffness affects the behavior of cells. [58][59][60][61] However, as an example of oral squamous cell carcinoma (ORCC), it exhibits a dual phenotype in which cells do not appear to exhibit cellular memory when cells initially cultured on stiff and re-cultured on soft matrix, while those re-cultured on a stiff matrix appear to be primed for migration. 62 Our results are consistent with theirs in which the EMT and associated signaling changes induced by stiff matrix were reversed when hela cell initially cultured on stiff and re-cultured on soft matrix, which shows cells did not appear to exhibit mechanical memory. Therefore, it would be important to further investigate the mechanical memory of cells and the role of Pin1/YAP in it.
In summary, the present study demonstrated from in vivo and in vitro experiments that matrix stiffness promotes EMT in cervical cancer, and the Pin1/YAP pathway plays an important role in this (Figure 6d). This may provide new ideas for the design of clinical drug targets.

| Cell culture
The human cervical cancer cell lines hela and hela-RFP were cultured in dulbecco's modified eagle medium (DMEM) medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Biological Industries) and 100 units/ml of penicillin/streptomycin (Hyclone). All cells were cultured at 37 C and 5% CO 2 (Thermo fisher scientific).

| Antibodies and reagents
Complete information on all antibodies and reagents used in this study are provided in Table S1 and S2.

| Immunofluorescence
IF was performed according to a method described previous. 63 The cells were fixed with 4% paraformaldehyde (Leagene) for 30 min at room temperature (RT), permeabilized with 0.1% TritonX-100 (Solarbio) for 20 min at room temperature and then closed with 5% BSA (Solarbio) for 20 min. Primary antibodies were incubated overnight at 4 C, followed by incubation in DyLight-coupled secondary antibodies for 1 h. Cytoskeleton staining was then incubated with FITC-labeled phalloidin (Yisheng Biotechnology) for 1 h. Nucleus were stained with DAPI (Solarbio). Immunofluorescence microscopy was observed for staining (Nikon Ti2).

| Preparation of PA hydrogels
Preparation of PA hydrogels was performed according to a method described previous. 22

| Animal studies
Cell-derived xenograft model was performed according to a method described previous. 27  After mice were operated, tumors were excised and frozen or fixed in paraformaldehyde in preparation for the next experiments.

| Total and nuclear-cytosol protein extraction and Western blot analysis
Total proteins were extracted from cells using RIPA lysis buffer containing protease inhibitors (Beyotime). Nuclear and cytoplasmic proteins were extracted from cells using the Nucleocytoplasmic Extraction Kit (Beyotime) according to the manufacturer's instructions. WB analysis was carried out as previously reported. 64 For westernblot analysis, primary antibody designation is shown in Table S1.

| Quantitative PCR analysis
Total RNA was extracted from treated cells using Trizol (Absin) and reverse transcribed into cDNA using the PrimeScriptRT kit (Takara).
Experiments were performed using faststart essential DNA green

| Masson staining and immunochemistry
Tumor tissues were fixed in 4% paraformaldehyde (Leagene), embedded in paraffin, and then sectioned (5 μm). For Masson's trichrome staining, a standard protocol was used to assess fibrosis. 65 For immunohistochemical analysis, 65 the primary antibody designation is shown in Table S1. These images were taken with a microscope (Nikon Ti2).

| Cell migration assay
First, the cells were inoculated in a six-well plate placed on hydrogel slides, partially covered by sterile slides so as to form a clear "edge" without physically harming the cell monolayer. Then incubated for 24 h to form a fused monolayer, after which the covered slides were carefully removed. Cells were rinsed twice with PBS and photographed under a microscope (Nikon Ti2). Subsequently, fresh DMEM containing 2% fetal bovine serum was added to six-well plate culture dishes and incubated in an incubator for 24 h. At last, the well plates were removed and photographed under the microscope to observe cell migration. The experiment was repeated three times under each treatment condition.
Quantitative assessment was performed using ImageJ software. The performed method was carried out as previously reported. 66

| Statistical analysis
All experiments were repeated thrice; numerical data are expressed as mean ± standard error (SEM), and statistical significance (defined as p < 0.05) was determined using the two-sample t-test of

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.